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Modulation of CFTR stability and function from the extracellular space

Final Report Summary - LOOP ABC (Modulation of CFTR stability and function from the extracellular space)

There are over 1500 CFTR mutations identified in the CFTR gene, and many of the mutations affect the biosynthetic maturation of the chloride channel. The main objective of the project is to characterise the long extracellular loop of CFTR that will establish the basis of design of hydrophilic drugs, which can affect the protein function and stability from the extracellular space.

As a first step, we structurally characterised the longer extracellular loops of CFTR. Obtaining high resolution structural data of large transmembrane proteins is extremely challenging; therefore, we aimed to determine the structure of isolated extracellular loops by computational and experimental methods. A large and diverse set of structures of the longest, fourth extracellular loop of CFTR were generated using discrete molecular dynamics (DMD) with an all-atom force field. Peptides corresponding to wild type and mutant sequences of EL4 have been synthesised and subjected to nuclear magnetic resonance (NMR) spectroscopy. Although the peptides were not labelled, the NOESY, TOCSY, and COSY NMR spectra allowed assignments of the observed peaks thus determine the secondary structural elements in the peptides. Although the secondary structure of conformations resulted from the ab initio folding simulations correlate with the experimental conformations, folding simulations constrained by data of NMR spectroscopy are being performed to gain more accurate structural information on the extracellular loop. The structure and dynamics of the wild type and mutant peptides will reveal the differences between the extracellular loops in CFTR mutants with altered function.

We hypothesise that changes in the conformation or dynamics of the extracellular loops alter the conformation of the transmembrane helices that changes the protein function. To test this hypothesis, we aimed to study the role of EL mutations in the dynamics of the transmembrane helices in intact CFTR. Effect of loop mutations on inter-helix distances is being measured as intramolecular cross-linking between transmembrane helices. Since the identification of amino acid pairs for cross-linking was not efficient and resulted in low number of construct with sufficient expression level, we collected mutations of other ABC proteins for comparative analysis aiding generation of mutants. We developed a framework to mine mutations from full text papers and a web application for their analysis (see http://abcmutations.hegelab.org for details). In parallel, we performed molecular dynamics (MD) simulations with full length ABC proteins embedded in a lipid bilayer to characterise the motions and different conformations of these proteins.

Results

1. The in silico folding of EL4 WT and Ins2 (a 6 a.a. insertion at a.a. 890, decreasing opening) exhibited stable structures, while EL4 Ins1 (a 10 a.a. insertion at a.a. 890 increasing channel opening) resulted in a diverse set of conformations. These results suggest that the increasing flexibility of EL4 promotes CFTR gating.
2. Based on essential dynamics analysis of simulations of the full-length human MDR1 model, we propose that the absence of nucleotides can drive the system towards a 'bottom-closed' apo conformation. We have also found significant structural instability of the 'bottom-open' form of the transporters in MD simulations. Based on these data, we suggest that the 'bottom-closed' conformation is physiologically more relevant for describing the structure of the apo state. Based on our results on dynamics of ABC transporters, we have been invited to contribute a review paper in Current Pharmacological Design.
3. We created a database and web application for comparative analysis of mutations in ABC proteins (see http://abcmutations.hegelab.org for details). These help not only our work but also researchers in the field - there are months with over 600 visits.
4. To prepare in silico docking to the EL4 structural model, we performed docking of drugs in clinical trials against cystic fibrosis, to CFTR. The docking locations are supported by experiments done in the laboratory of our collaborator, Gergely Lukacs (McGill University, Montreal, Canada). Our docking results together with experimental validation strongly suggest that structure-based combination selection strategy can robustly rescue the conformationally defective mutant dF508 CFTR.

The structural properties of the fourth extracellular loop will provide a solid basis to design drugs to stabilise the functional form of CFTR. The general applicability of our idea and the employed computational tools to other disease-causing ABC proteins are also planned to be tested on multi-drug transporters. Our results indicate that impaired function of CFTR mutants may be rescued by increasing the flexibility of EL4. In contrast, when the objective is the inhibition of a specific ABC protein, such as a multi-drug transporter, an increase in the rigidity of the analogous extracellular loop is desired.

Socio-economic impacts

Dr Hegedus, moved back to Hungary in 2009 and got the possibility to build his own research group from the beginning of 2010. He was offered a 5 year long position in the Molecular Biophysics Research Group of the Hungarian Academy of Sciences staring July of 2012. However, this Marie Curie Action does not simply act against the brain-drain of the United States of America (USA) helping in the reintegration of Dr Hegedus, but also contributed to create job opportunities for three other persons increasing research capacity in Hungary: a new laboratory with novel techniques and ideas was established.

The laboratory trains scientists for the future, as it is involved in both undergraduate and graduate courses. One of the members successfully completed his PhD, while another member started her PhD studies. One undergraduate student from the Budapest University of Technology and Economics completed his thesis in the lab, and now three undergraduate students from the School of Information Technology, Pazmany Peter Catholic University and Budapest University of Technology and Economics perform their practical work for their diploma in the principal investigator (PI)'s laboratory. Dr Hegedus serves at PhD comities, reviews manuscripts and grant applications for both national and international publishers and agencies. He has also been invited to the scientific board of the 2013 National Membrane Transport Conference, Sumeg, Hungary.

To promote European reintegration, Dr Hegedus visited Oxford University (United Kingdom (UK)) to initiate collaborations (computational biology, Mark Sansom; NMR spectroscopy, Christina Redfield). He has started collaborations with Amos Bairoch (Swiss Institute of Bioinformatics, Geneva, Switzerland) and Kaspar Locher (ETH, Zurich, Switzerland). In parallel, he maintains active oversee interactions with Nikolay V. Dokholyan (UNC, Chapel Hill, USA), Daniel M. Zuckerman (University of Pittsburgh, Pittsburgh, Palo Alto, USA), and Gergely Lukacs (McGill University, Montreal, Canada).

Contact details

tamas@hegelab.org
http://www.hegelab.org